The long-term goal of this research program is to determine the sequence of molecular events during cellular duplication and the chemical mechanisms which regulate the initiation and completion of each of the major events in the sequence. The major objectives of the current project are the determination of the molecular mechanisms which control the initiation of DNA replication and the initiation of cell division in Escherichia coli. Four projects will be undertaken in the next year. 1) We suggest that the timing of initiation of chromosome replication in E. coli B/r is determined by elongation of the bacterial envelope between the membrane-bound origins of sister chromosomes. To test this model, the effect of membrane stretching on initiation of chromosome replication will be assayed by investigating the effects of swelling in spheroplasts and sucrose-dependent fragile mutants. 2) The relationship between chromosome replication and cell division in three different lines of E. coli B/r will be investigated by pulse-labeling exponetially growing cultures growing at different rates with C14- thymidine and measuring the rate of thymidine incorporation during the division cycle with the membrane elution technique. Parameters to be measured are C, D, C plus D, G1 and cell size in cells growing with interdivision times between 25 min and 12 hr. 3) The coordination between F' lac replication and chromosome replication, and the requirements for RNA and protein synthesis to trigger F' lac replication, will be investigated in synchronously dividing cultures of the three lines of E. coli B/r. 4) The rate of synthesis and pattern of segregation of membrane phospholipids will be analyzed in synchronous cultures of E. coli B/r.